Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.14/182693
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- Title
- Determination of site-specific glycan heterogeneity on glycoproteins
- Related
- Nature protocols, Vol. 7, No. 7, (2012), p.1285-1298
- DOI
- 10.1038/nprot.2012.062
- Publisher
- Nature Publishing
- Date
- 2012
- Author/Creator
- Kolarich, Daniel
- Author/Creator
- Jensen, Pia H
- Author/Creator
- Altmann, Friedrich
- Author/Creator
- Packer, Nicolle H
- Description
- The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography- electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO a nd IgG, described in the second protocol of this series (10.1038/nprot.2012. 063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides.
- Description
- 14 page(s)
- Resource Type
- journal article
- Organisation
- Macquarie University. Biomolecular Frontiers Research Centre
- Identifier
- http://hdl.handle.net/1959.14/182693
- Identifier
- ISSN:1754-2189
- Identifier
- mq_res-ext-2-s2.0-84862238705
- Language
- eng
- Reviewed
